The white blood cell (WBC) differential is one component of the complete blood count (CBC) which can deliver information on a variety of medical conditions including infection, allergic reactions and drug responses. A WBC differential cannot typically be performed at the point-of-care or in an emergency setting, necessitating transfer to an off-site centralized facility in order to be analyzed on conventional hematology analyzers. State-of-the-art analyzers, while highly accurate and capable of simultaneously determining a large number of parameters relating to erythrocytes, leukocytes and platelets, possess a large footprint, require dedicated staff and rely on expensive reagents. Efforts are currently being directed at developing portable hematology analyzers based on label-free technologies that could perform WBC differentials at the point-of-care or in situations where a rapid and accurate analysis is more critical than a more comprehensive but delayed evaluation. Various methods are being explored to engineer such low-cost analyzers using intrinsic cellular parameters such as size, impedance and dielectric properties as separation criteria. Lateral di-electrophoresis and hydrodynamic separation are two recently developed microfluidic techniques, which can separate a particle flow into different size-dependent flows. Both techniques have been used to separate whole blood into platelets, erythrocytes and leukocytes. However, without actively altering the size of either monocytes or granulocytes, a leukocyte differential has remained difficult to achieve. Alternatively, generating a 3-part WBC differential was shown to be possible with impedance spectroscopy. Cell size and internal composition are translated into characteristic impedance signals measurable by electrodes positioned in a microfluidic channel. By performing a dual frequency analysis a 3-part classification of the main leukocyte subtypes was achieved. Lens-free in-line holographic microscopy has emerged as a promising label-free, cell-analysis technique which delivers a cell image by capturing the interference pattern of scattered and transmitted light of a cell directly on a CMOS chip, in the absence of objective lenses and other complex optics. Software-based reconstruction of the interference pattern generates an image of the cell, which retains its morphological signature. Given the relative simplicity of its optical components, lens-free microscopy holds great potential for miniaturization and integration into a microfluidic blood analysis platform that could be used at the point-of-care or in emergency settings.
This technique has already been shown to be compatible with a variety of biological specimens, including blood cells. Wide field-of-view lens-free holographic microscope have been used to capture holograms of cells within a diluted blood sample and showed the reconstructed images of erythrocytes, leukocytes and platelets to be comparable to images captured with a conventional microscope equipped with a 40× objective-lens. Holograms of Wright-Giemsa-stained blood smears were acquired and used the recovered phase and amplitude images to discriminate between the three main leukocyte populations. Albeit not label-free, these measurements pointed to the potential of generating a 3-part WBC differential based on analysis of holographic images. In all these in-line holographic geometries, a large field-of-view configuration is exploited by using plane wave illumination. Reconstructions of holograms taken with a plane wave holographic geometry are typically limited by the camera's pixel pitch. This limit can be overcome by taking multiple images under slightly different angles of the illumination source or with a subpixel shift of the sample or camera. While this configuration is not compatible with imaging fast moving objects such as cells owing in a microfluidic channel, subpixel resolution can still be achieved by using point-source illumination placed close to the object. The spherical wavefront of the point source serves as an at-lens transformation, magnifying the image and increasing the resolution. This point-source, digital, in-line, holographic microscopy (PSDIHM) geometry has been used to image aquatic organisms and to obtain detailed phase information of various cell types.